Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, which encodes for an apical membrane epithelial protein that functions as a regulator of several channels, including the c-AMP-regulated chloride channel. As a result, chloride transfer across the epithelial membranes of CF patients is abnormal.
While CF symptoms appear in a number of organ systems, including the respiratory, gastrointestinal, and reproductive tract, for most patients, the most important pathological changes associated with the CFTR defect are observed in the lungs. Patients with suffering from CF produce excessive quantities of abnormally viscous mucus, which blocks the patient's bronchi and readily becomes infected. As a result, CF patients are stricken with chronic respiratory infections, including Pseudomonas infections, causing inflammation, progressive airway damage, and bronchiectasis. Pulmonary complications are the primary cause of CF-related morbidity and mortality in CF patients. Although improved treatment of lung disease has increased survival in CF patients, the median predicted age for survival for CF patients is only 35 years. In addition, CF patients continue to have significant morbidity, including frequent hospitalizations.
Mutations in a single gene—the Cystic Fibrosis Transmembrane Regulator (CFTR) gene—causes CF. The gene was discovered in 1989. Since then, more than 900 mutations of this single gene have been identified. In normal cells, the CFTR protein acts as a channel that allows cells to release chloride and other ions. But in people with CF, this protein is defective and the cells do not release the chloride. The result is an improper salt balance in the cells and thick, sticky mucus. Researchers are focusing on ways to cure CF by correcting the defective gene, or correcting the defective protein. Aerosol gene therapy is the most direct therapeutic strategy for CF lung diseases. Since the discovery of the CFTR gene in 1989, a large number of gene carrier, including both viral and non-viral systems, have been developed and tested in the lungs of CF patients (Griesenbach, U. and Alton, E. W. F. W. Adv. Drug Deliv. Rev. 61:128-139 (2009)).
As described by Flotte, et al., in CHEST 120(3 suppl) 124S-131S (2001), cystic fibrosis transmembrane conductance regulator (CFTR) gene replacement can decrease morbidity and mortality from cystic fibrosis (CF). In vivo gene transfers have been accomplished in CF patients. Choice of vector, mode of delivery to airways, translocation of genetic information, and sufficient expression level of the normalized CFTR gene are issues that limit efficacy. Initial studies with adenovirus (Ad) vectors resulted in a vector that was efficient for gene transfer with dose-limiting inflammatory effects due to the large amount of viral protein delivered. The next generation of Ad vectors, with more viral coding sequence deletions, has a longer duration of activity and elicits a lesser degree of cell-mediated immunity in mice. A more recent generation of Ad vectors has no viral genes remaining. Despite these changes, the problem of humoral immunity remains with Ad vectors. A variety of strategies such as vector systems requiring single, or widely spaced, administrations, pharmacologic immunosuppression at administration, creation of a stealth vector, modification of immunogenic epitopes, or tolerance induction have been considered to circumvent humoral immunity. The level of CFTR messenger RNA expression is difficult to ascertain with adeno-associated virus (AAV) vectors since the small size of the vector relative to the CFTR gene leaves no space for vector-specific sequences on which to base assays to distinguish endogenous from vector-expressed messenger RNA. In general, AAV vectors appear to be safer and have superior duration profiles, but do not elimination the problems with immune responses. Another challenge to the development of clinically feasible gene therapy is delivery mode. Early pulmonary delivery systems relied on the direct instillation of aerosolized vectors, which can result in the induction of adverse reactions because vector is delivered into the lung parenchyma. More recent studies have examined the potential for using spray technologies to target aerosolized AAV vectors to the larger central airways, thereby avoiding alveolar exposure and adverse effects. Other modes of delivery, such as cationic liposomes, lipid-DNA complexes, generally have been less efficient than viral vectors but do not stimulate inflammatory and immunologic responses.
Despite many years of research efforts and investment, there has been little success with CF gene therapy to date. The failures of gene therapies for CF have been attributed to a number of biological barriers, including limited cellular uptake across the apical membrane (Jiang, G. et al. Hum. Gene. Ther. 9:1531-1542 (1998), Matsui, H. et al., J. Biol. Chem. 272:1117-1126 (1997)), unproductive intracellular trafficking (Ferrari, S. et al. Adv. Drug Deliv. Rev. 54:1373-1393 (2002)), carrier toxicity, and immunogenicity (Ferrari, S. et al. Clin. Exp. Immunol. 132:1-8 (2003). A largely overlooked barrier to effective gene therapies for CF patients is the highly adhesive and hyperviscoelastic CF sputum that can immobilize and trap gene carriers, thereby greatly reducing the flux of gene carriers that can reach the airway epithelium, or exclude them altogether (Lai, S. K. et al. Adv. Drug Deliv. Rev. 61:158-171 (2009), Sanders, N. N. et al. Am. J. Respir. Crit. Care Med. 162:1905-1911 (2000), Suk, J. S. et al. Biomaterials. 30:2591-2597 (2009)).
CF sputum is composed of a dense mesh of mucin fibers, large macromolecules containing a high density of negatively charged glycans interspersed with periodic hydrophobic regions (Lai, S. K. et al. Adv. Drug Deliv. Rev. 61:158-171 (2009), Cone, R. A. Adv. Drug Deliv. Rev. 61:75-78 (2009)). Elevated levels of bacterial and endogenous DNA, as well as actin filaments from degraded neutrophils in CF sputum, further contribute to its dense mesh structure and increased adhesivity (Sanders, N. et al. Adv. Drug Deliv. Rev. 61:115-127 (2009), Voynow, J. A. and Rubin, B. K. Chest. 135:505-512 (2009)). The average pore size in CF sputum has been estimated at 145±50 nm (range: 60-300 nm) (Suk, J. S. et al. Biomaterials. 30:2591-2597 (2009)), markedly smaller than the 340±70 nm for healthy human mucus secretions (Lai, S. K. et al. Proc. Natl. Acad. Sci. U.S.A. 107:598-603 (2010)). As a consequence of the elevated adhesivity and tighter mesh size, existing viral (Hida, K. et al. PLoS ONE. 6:e19919 (2011)) and non-viral (Suk, J. S., et al. Mol. Ther. 19:1981-1989 (2010)) gene carriers are extensively immobilized within CF sputum, suggesting that the inability to penetrate CF sputum may explain the limited success of gene therapies for CF.
In order to successfully deliver genetic therapies to the lungs of CF patients and patients with other lung diseases such as asthma, COPD and lung cancers, or other regions having mucus barriers to entry such as the gastrointestinal and reproductive tracts, gene carriers which can penetrate highly adhesive mucus and not elicit an immune response are required.
Therefore, it is an object of the invention to provide vehicles for efficient gene delivery through mucus by improved diffusion through the mucus without eliciting significant immunogenicity.